Felix Kurth

A new mechanobiological era: microfluidic pathways to apply and sense forces at the cellular level.

Fueled by technological advances in micromanipulation methodologies, the field of mechanobiology has boomed in the last decade. Increasing needs for clinical solutions to better maintain our major mechanosensitive tissues (muscle, bone, and cartilage) with increasing age and new insights into cellular adaptations to mechanical stresses beckon for novel approaches to meet the needs of the future. In particular, the emergence of microfluidics has inspired new interdisciplinary strategies to decipher cellular mechanotransduction on the biochemical as well as macromolecular level. Cellular actuation by locally varying fluid shear can serve to accurately alter membrane surface tension as well as produce direct compressive and strain forces onto cells. Moreover, incorporating microelectronic technologies into microfluidic platforms has led to further advances in actuation and readout possibilities. In this review, we discuss the application of microfluidics to mechanobiological research with particular focus on microfluidic platforms that are able to simultaneously monitor cellular adaptation to mechanical forces and interpret biochemical mechanotransduction.

Single cells in confined volumes: microchambers and microdroplets.

Microfluidic devices capable of manipulating and guiding small fluid volumes open new methodical approaches in the fields of biology, pharmacy, and medicine. They have already proven their extraordinary value for cell analysis. The emergence of microfluidic platforms has paved the way to novel analytical strategies for the positioning, treatment and observation of living cells, for the creation of chemically defined liquid environments, and for tailoring biomechanical or physical conditions in small volumes. In this article, we particularly focus on two complementary approaches: (i) the isolation of cells in small chambers defined by microchannels and integrated valves and (ii) the encapsulation of cells in microdroplets. We review the advantages and limitations of both approaches and discuss their potential for single-cell analysis and related fields. Our intention is also to give a recommendation on which platform is most appropriate for a new question, i.e., a guideline to choose the most suitable platform.

On-chip enzyme quantification of single Escherichia coli bacteria by immunoassay-based analysis.

Individual bacteria of an isogenic population can differ significantly in their phenotypic characteristics. This cellular heterogeneity is thought to increase the adaptivity to environmental changes on a population level. Analytical methods for single-bacteria analyses are essential to reveal the different factors that may contribute to this cellular heterogeneity, among them the stochastic gene expression, cell cycle stages and cell aging. Although promising concepts for the analysis of single mammalian cells based on microsystems technology were recently developed, platforms suitable for proteomic analyses of microbial cells are by far more challenging. Here, we present a microfluidic device optimized for the analysis of single Escherichia coli bacteria. Individual bacteria are captured in a trap and isolated in a volume of only 155 pL. In combination with an immunoassay-based analysis of the cell lysate, the platform allowed the selective and sensitive analysis of intracellular enzymes. The limit of detection of the developed protocol was found to be 200 enzymes. Using this platform, we could investigate the levels of β-galactosidase in cells grown under different nutrient conditions. We successfully determined the enzyme copy numbers in cells cultured in defined medium (3517 ± 1578) and in complex medium (4710 ± 2643), and verified the down-regulation of expression in medium that contained only glucose as carbon source. The strong variations we found for individual bacteria confirm the phenotype heterogeneity. The capability to quantify proteins and other molecules in single bacterial lysates is encouraging to use the new analysis platform in future proteomics studies of isogenic bacteria populations.

Transient receptor potential vanilloid 2-mediated shear-stress responses in C2C12 myoblasts are regulated by serum and extracellular matrix

The developmental sensitivity of skeletal muscle to mechanical forces is unparalleled in other tissues. Calcium entry via reputedly mechanosensitive transient receptor potential (TRP) channel classes has been shown to play an essential role in both the early proliferative stage and subsequent differentiation of skeletal muscle myoblasts, particularly TRP canonical (TRPC) 1 and TRP vanilloid (TRPV) 2. Here we show that C2C12 murine myoblasts respond to fluid flow‐induced shear stress with increments in cytosolic calcium that are largely initiated by the mechanosensitive opening of TRPV2 channels. Response to fluid flow was augmented by growth in low extracellular serum concentration (5 vs. 20% fetal bovine serum) by greater than 9‐fold and at 18 h in culture, coincident with the greatest TRPV2 channel expression under identical conditions (P < 0.02). Fluid flow responses were also enhanced by substrate functionalization with laminin, rather than with fibronectin, agreeing with previous findings that the gating of TRPV2 is facilitated by laminin. Fluid flow‐induced calcium increments were blocked by ruthenium red (27%) and SKF‐96365 (38%), whereas they were unaltered by 2‐aminoethoxydiphenyl borate, further corroborating that TRPV2 channels play a predominant role in fluid flow mechanosensitivity over that of TRPC1 and TRP melastatin (TRPM) 7.—Kurth, F., Franco‐Obregón, A., Casarosa, M., Küster, S. K., Wuertz‐Kozak, K., Dittrich, P. S. Transient receptor potential vanilloid 2‐mediated shear‐stress responses in C2C12 myoblasts are regulated by serum and extracellular matrix. FASEB J. 29, 4726‐4737 (2015). www.fasebj.org

Bilayer microfluidic chip for diffusion-controlled activation of yeast species.

A bilayer microfluidic chip is used, in which multiple laminar streams are generated to define local microenvironments. The bilayer architecture of the microchip separates cell handling and positioning from cell activation by soluble chemicals. Cell activation is diffusion controlled through a porous membrane. By employing time-lapse fluorescence microscopy, gene expression of the enhanced green fluorescent protein (eGFP) in Saccharomyces cerevisiae is studied under various conditions. We demonstrate that the yeast cells remain viable in the microchip for at least 17 h, and that gene expression can be initiated by the supply of the inducer galactose at a spatial precision of a few micrometers.

Cell Penetration, Herbicidal Activity, and in-vivo-Toxicity of Oligo-Arginine Derivatives and of Novel Guanidinium-Rich Compounds Derived from the Biopolymer Cyanophycin

Oligo-arginines are thoroughly studied cell-penetrating peptides (CPPs, Figures 1 and 2). Previous in-vitro investigations with the octaarginine salt of the phosphonate fosmidomycin (herbicide and anti-malaria drug) have shown a 40-fold parasitaemia inhibition with P. falciparum, compared to fosmidomycin alone (Figure 3). We have now tested this salt, as well as the corresponding phosphinate salt of the herbicide glufosinate, for herbicidal activity with whole plants by spray application, hoping for increased activities, i.e. decreased doses. However, both salts showed low herbicidal activity, indicating poor foliar uptake (Table 1). Another pronounced difference between in-vitro and in-vivo activity was demonstrated with various cell-penetrating octaarginine salts of fosmidomycin: intravenous injection to mice caused exitus of the animals within minutes, even at doses as low as 1.4 μmol/kg (Table 2). The results show that use of CPPs for drug delivery, for instance to cancer cells and tissues, must be considered with due care. The biopolymer cyanophycin is a poly-aspartic acid containing argininylated side chains (Figure 4); its building block is the dipeptide H-βAsp-αArg-OH (H-Adp-OH). To test and compare the biological properties with those of octaarginines we synthesized Adp8-derivatives (Figure 5). Intravenouse injection of H-Adp8-NH2 into the tail vein of mice with doses as high as 45 μmol/kg causes no symptoms whatsoever (Table 3), but H-Adp8-NH2 is not cell penetrating (HEK293 and MCF-7 cells, Figure 6). On the other hand, the fluorescently labeled octamers FAM-(Adp(OMe))8-NH2 and FAM-(Adp(NMe2))8-NH2 with ester and amide groups in the side chains exhibit mediocre to high cell-wall permeability (Figure 6), and are toxic (Table 3). Possible reasons for this behavior are discussed (Figure 7) and corresponding NMR spectra are presented (Figure 8).

Influence of the Membrane Dye R18 and of DMSO on Cell Penetration of Guanidinium-Rich Peptides

A quantitative analysis by confocal fluorescence microscopy of the entry into HEK293 and MCF‐7 cells by fluorescein‐labeled octaarginine (1) and by three octa‐Adp derivatives (2 – 4, octamers of the β‐Asp‐Arg‐dipeptide, derived from the biopolymer cyanophycin) is described, including the effects of the membrane dye R18 and of DMSO on cell penetration.